Haemophilus influenzae (Hi) is a gram-negative coccobacillus and a strict human commensal. Strains of Hi are either encapsulated in a polysaccharide capsule or are non-encapsulated and are accordingly classified into typeable (encapsulated) and non-typeable (non-encapsulated) strains.
Encapsulated pathogenic strains of Hi cause mainly, but not exclusively, invasive disease in children under six years of age. Haemophilus influenzae type b (Hib), for example, is a major cause of meningitis and other invasive infections in children. Effective vaccines exist against Hib infections, and are based on producing antibodies to the polysaccharide capsule, and are therefore ineffective against non-typeable Haemophilus influenzae (ntHi).
Non-typeable Haemophilus influenzae (ntHi) represents the majority of the colonising strains and, although rarely invasive, are responsible for a significant proportion of mucosal disease including otitis media, sinusitis, chronic conjunctivitis and chronic or exacerbation of lower respiratory tract infections. Currently, approximately 30%, and as much as 62% of ntHi are resistant to penicillins. Carriage is estimated at 44% in children and approximately 5% in adults, and can persist for months. Neither the pathogenic mechanisms nor the host immunological response has been fully defined for otitis media caused by ntHi.
Otitis media is a common disease in children less than 2 years of age. It is defined by the presence of fluid in the middle ear accompanied by a sign of acute local or systemic illness. Acute signs include ear pain, ear drainage, hearing loss whereas systemic signs include fever, lethargy, irritability, anorexia, vomiting or diarrhoea. Streptococcus pneumoniae and non-typeable Haemophilus influenzae (nthi) are the most predominant bacteria that cause the condition, accounting for 25-50%, and 15-30% of the species cultured, respectively. Moraxella catarrhalis is another common cause of the disease. In addition, ntHi is responsible for 53% of recurrent otitis media. Approximately 60% and 80% of children have at least one episode of the disease by 1 and 3 years of age respectively (the peak being around 10 months).
There is evidence that protective immunity does exist for ntHi, however antigenic drift in the epitopes naturally involved (outer-membrane proteins P2, P4, P6) plays a major role in the ability of ntHi to evade the immune defence of the host.
There is therefore a need for additional effective vaccines against Haemophilus influenzae, and particularly for vaccines against non-typeable Haemophilus influenzae which is not affected by the currently available Hi polysaccharide vaccines.
Major Outer Membrane Protein (MOMP) P5 is a heat-modifiable outer membrane protein of H. influenzae. P5 may play a role in ntHi pathogenesis as an adhesin by binding to respiratory mucin or to RSV-infected respiratory epithelial cells (Reddy et al. (1996) Infect. Immun. 64:1477-1479; Jiang et al. (1999) Infect. Immun. 65:1351-1356). This binding activity could be mediated by surface exposed regions of the protein. The protein has been shown to be a protective antigen in various models.
There are conflicting reports with regards to the structure of this protein. Although it has been reported that the protein adopts a fimbriae structure composed of assembled coiled coils, this is contradictory to the similarity of sequences observed between P5 and E. coli OmpA which is an eight-stranded β-barrel-forming protein with four surface exposed loops (Munson et al. (1993) Infect Immun. 61:4017-4020).
During persistent infections by ntHi in patients with chronic bronchitis, ntHi variant strains with alterations in their OMP P5 sequences appear. Also, isolates from different anatomical sites display such variability. However this variability is mostly limited to 4 regions. These regions correspond to the regions predicted as surface exposed and as a consequence that could be exposed to the immune system pressure. Upon infection, the appearance of P5 strain variants could be an escape mechanism for ntHi or could enable the bacteria to colonise different anatomical sites (Webb and Cripps (1998) J. Med. Microbiol. 47:1059-1067; Duim et al. (1997) Infect. Immun. 65:1351-1356). Even so, it has been shown that mice anti-P5 purified antibodies were bactericidal for the homologous and a few heterologous ntHi strains (Quigley-Reape et al. (1995) Abstr. E70, p 239. In Abstracts of the 95th ASM general meeting 1995).
LB1(f) is a 19 amino-acid peptide derived from the sequence of MOMP P5 from strain ntHi1128 (occupying the region Arg117 to Gly135). This peptide was defined as being the third exposed loop of P5, and as being a potential B cell epitope, by analysis of the primary sequence of P5. Immunising animals with chimeric fimbrin peptides (called LB1 peptides), comprising: the LB1(f) peptide; a linker peptide; and a T cell epitope, induces a protective immune response to the MOMP P5 and reduces the colonization of ntHi in animals subsequently exposed to ntHi (see U.S. Pat. No. 5,843,464).
The problem with using protein antigens from only one strain of H. influenzae in a vaccine is that protection conferred tends to be largely restricted to homologous challenge [Bakaletz et al. (1997) Vaccine 15:955-961; Haase et al. (1991) Infect. Immun. 59:1278-1284; Sirakova et al. (1994) Infect. Immun. 62:2002-2020]. The antigenic diversity of the ntHi Outer Membrane Proteins, means that development of a broadly effective vaccine against a group of organisms as heterogeneous as ntHi will require a new strategy.
WO 99/64067 discloses a more effective use of the LB1(f) peptide as a vaccine against a broad spectrum of heterologous Haemophilus influenzae strains that express the MOMP P5 (or naturally occurring variants of the protein). This involved the identification of the 3 antigenic groups of LB1(f) peptides that define the population of LB1(f) peptides present in heterologous ntHi MOMP P5 proteins, Chimeras of these peptides were suggested as a immunogen in order to obtain a protective immune response against a large variety of ntHi strains.
A problem that exists is that in order for these peptides to work optimally as effective immunogens, they must be able to generate antibodies which recognise and bind to the epitopes in their native structure.
Accordingly, the present invention relates to a method of increasing the effectiveness of the LB1(f) peptides by inserting them into surface exposed loops of other outer membrane proteins, or, preferably, back into MOMP P5 itself such that the epitopes may be better recognised in their native conformation by the immune system. Such recombinant outer membrane proteins of the invention have one or more of the following advantages: the context of the important LB1(f) B epitopes is in a favourable context (with a constrained structure) for better immune recognition and immunogenicity; protective LB1(f) epitopes may replace hypervariable, non-protective epitopes from the outer membrane protein to focus the immune response to the protective LB1(f) peptides; the recombinant, modified outer membrane protein helps to provides a better protective immune response against a wide range of ntHi strains.